System and method for monitoring cellular activity

ABSTRACT

A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/817,297, filed Apr. 2, 2004, which is a continuation of U.S.application Ser. No. 10/159,703, filed May 28, 2002, which is acontinuation of U.S. application Ser. No. 09/628,219, filed Jul. 28,2000, which issued as U.S. Pat. No. 6,403,332, on Jun. 11, 2002, whichclaims priority to U.S. Provisional Application Nos. 60/146,490, filedJul. 30, 1999 and 60/164,504, filed Nov. 9, 1999, the disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the monitoring of cellular activitythrough the use of excitable markers. More particularly, the inventionrelates to a system and method for using a plurality of fluorescentprobes to monitor cellular activity.

BACKGROUND OF THE INVENTION

Presently, fluorescence microscopy is one of the most widely usedmicroscopy techniques, as it enables the molecular composition of thestructures being observed to be identified through the use offluorescently-labeled probes of high chemical specificity, such asantibodies. However, its use is mainly confined to studies of fixedspecimens because of the difficulties of introducing antibody complexesinto living specimens. For proteins that can be extracted and purifiedin reasonable abundance, these difficulties can be circumvented bydirectly conjugating a fluorophore to a protein and introducing thisback into a cell. It is believed that the fluorescent analogue behaveslike the native protein and can therefore serve to reveal thedistribution and behavior of this protein in the cell.

An exciting, new development in the use of fluorescent probes forbiological studies has been the development of the use of naturallyfluorescent proteins as fluorescent probes, such as green fluorescentprotein (GFP). The gene for this protein has been cloned and can betransfected into other organisms, This can provide a very powerful toolfor localizing regions in which a particular gene is expressed in anorganism, or in identifying the location of a particular protein. Thebeauty of the GFP technique is that living, unstained samples can beobserved. There are presently several variants of GFP which providespectrally distinct emission colors.

Conventionally, fluorescence microscopy only worked well with very thinspecimens or when a thick specimen was cut into sections, becausestructures above and below the plane of focus gave rise to interferencein the form of out-of-focus flare. However, this can be overcome byoptical sectioning techniques, such as multi-photon fluorescencemicroscopy.

Multi-photon fluorescence microscopy involves the illumination of asample with a wavelength around twice the wavelength of the absorptionpeak of the fluorophore being used. For example, in the case offluorescein which has an absorption peak around 500 nm, 1000 nmexcitation could be used. Essentially no excitation of the fluorophorewill occur at this wavelength. However, if a high peak-power, pulsedlaser is used (so that the mean power levels are moderate and do notdamage the specimen), two-photon events will occur at the point offocus. At this point the photon density is sufficiently high that twophotons can be absorbed by the fluorophore essentially simultaneously.This is equivalent to a single photon with an energy equal to the sum ofthe two that are absorbed. In this way, fluorophore excitation will onlyoccur at the point of focus (where it is needed) thereby eliminatingexcitation of the out-of-focus fluorophore and achieving opticalsectioning.

Often, multiple fluorophores are used, with each fluorophore having adifferent spectra, some of which may overlap. Typically, the ability todistinguish between the respective fluorophores is only possible wherethe excitation spectra are separated, or where the fluorescencelifetimes are distinct.

Another approach is to selectively excite different fluorophores byusing various excitation photon wavelengths, each of which willapproximate the wavelength of the absorption peak of a correspondingfluorophore. Such an approach is not practical for a number of reasons.Firstly, it is difficult to rapidly tune the excitation wavelength ofthe laser providing the excitation photons. Secondly, there is typicallya very broad excitation spectrum, so that such an approach requires asignificant amount of time.

Thus, the need exists for a system and method for efficiently monitoringa plurality of fluorescent probes, and to record emissions spectrarelating to those probes for subsequent analysis. The present inventionaddresses these needs.

SUMMARY OF THE INVENTION

Briefly, the present invention is directed to a system and method formonitoring cellular activity in a cellular specimen. According to oneillustrative embodiment of the invention, a plurality of excitablemarkers are applied to the specimen. A multi-photon laser microscope isprovided to excite a region of the specimen and cause fluorescence to beradiated from the region. The radiating fluorescence is processed by aspectral analyzer to separate the fluorescence into wavelength bands.The respective fluorescence bands are then collected by an array ofdetectors, with each detector receiving a corresponding one of thewavelength bands.

According to another embodiment, the invention is directed to a systemfor monitoring cellular activity in a cellular specimen that contains aplurality of excitable markers. The system includes a laser microscopethat is operative to excite the markers in a region of the specimen, sothat those markers in the region radiate fluorescence. The system alsoincludes a tunable filter that is operative to process the fluorescenceand to pass a portion of the fluorescence wavelengths radiated by themarkers. The system still further includes a detector that is operativeto receive the processed fluorescence wavelengths.

In still another embodiment, the invention is directed to a system formonitoring cellular activity, including a two-photon laser microscopethat is operative to excite the markers in a region of the specimen suchthat the markers in the region radiate fluorescence. The system alsoincludes a detector that is operative to receive non-descannedfluorescence from the specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features and advantages of the present invention will bebetter understood by reference to the following detailed descriptionwhen considered conjunction with the accompanying drawings wherein:

FIG. 1 is a schematic diagram of a first illustrative embodiment of thepresent invention;

FIG. 2 is a schematic diagram that illustrates one embodiment of aspectral analyzer included in the system of FIG. 1;

FIG. 3 is a schematic diagram that illustrates a system according toanother illustrative embodiment of the invention;

FIG. 4 is a schematic diagram that illustrates yet another illustrativeembodiment of the invention;

FIG. 5 is a schematic diagram that illustrates still another embodimentof the invention;

FIG. 6 is a flow chart that depicts the operational flow of processingsoftware to process spectral data received by the systems of any ofFIGS. 1 through 5 according to one illustrative embodiment of theinvention;

FIGS. 7 a-c depict emissions spectra for various fluorescent dyes; and

FIG. 7 d depicts a measured spectrum from a specimen that containsplural different dyes.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, a novel system 10 is disclosed for capturingspectral data from a specimen 12 to which a plurality of excitablemarkers have been applied. In one embodiment, at least two fluorescentdyes (i.e., fluorescent probes) are applied to the specimen. Thefluorescent dyes have respective emission spectra that may or may notoverlap. In the case where the emission spectra are very similar, itbecomes difficult to separate out the emission spectra to determine thecontributions of each of the fluorescent dyes to the observed spectra.System 10 facilitates making such determinations in an efficient,reliable manner.

As shown in FIG. 1, system 10 includes a laser 14 that generates laserlight. The light emitted by laser 14 is focused by a lens 16 onto ashort pass dichroic mirror 18. As is well known, the dichroic mirror 18selectively reflects light according to its wavelength. Thus, thedichroic mirror is selected such that it reflects the light emitted bylaser 14.

The reflected light from dichroic mirror 18 is directed toward scanningoptics, shown schematically at 20. The scanning optics may include acontrollable deflection unit, or any other suitable structure thatallows the reflected light to be redirected for scanning of the specimen12 by the laser light, as is well known to those skilled in the art.

The directed light from scanning optics 20 is imaged by a microscopelens 22 onto or into the specimen 12. The laser light excites thefluorescent dyes in the region where the light is directed, such thatthose dyes fluoresce and emit light having respective emissions spectra.

In one embodiment, laser 14 is a two-photon laser. As is well known, atwo-photon laser microscope depends on the two-photon effect, by whichthe fluorescent dyes are excited not by a single photon, but rather bytwo relatively low energy photons that are absorbed contemporaneously bya fluorescent dye. Thus, the requirement for two coincident (or nearcoincident) photons to achieve excitation of the fluorescent dye meansthat only focused light reaches the required intensities and thatscattered light does not cause excitation of the fluorescent dyes. Assuch, a two-photon laser microscope is inherently insensitive to theeffects of light scattering in thick slices. Alternatively, the laser 14may comprise some other multi-photon laser, or a single-photon laser.

The fluorescence radiated by the excited dyes is focused by themicroscope lens 22, and passes through the scanning optics 20 anddichroic mirror 18. The fluorescence is then focused by a lens 24 anddirected to a light guide 26 that delivers the focused light to aspectral analyzer 28. Spectral analyzer 28 receives the light anddisperses the light into respective wavelength bands. Each band isdetected by a corresponding detector, and the intensity of each band isrecorded and processed to determine the ingredients of the receivedfluorescence, as is described in more detail below in connection withFIG. 2.

Referring to FIG. 2, there is shown one illustrative embodiment ofspectral analyzer 28. Spectral analyzer 28 includes a housing 30 formedwith a light entrance opening 32 and a light exit opening 34. Lightentrance opening 32 is connected to light guide 26 to receive light fromthe light guide. Preferably, the distal end of light guide 26 includesan exit slit 27 to permit the delivery of fluorescence from light guide26 to spectral analyzer 28. Mounted within spectral analyzer 28 is afirst mirror 36 that directs the incoming light to a grating 38, whichdisperses the light over appropriate wavelength range. The dispersedlight is then directed to a second mirror 40 that directs the dispersedlight through the exit opening 34. In place of the grating 38, spectralanalyzer 28 may alternatively include a prism or other light-dispersingstructure.

Aligned with exit opening 34 is a detector array 42 consisting ofindividual detectors 43. The detector array 42 may take many differentforms, such as an array of photomultiplier tubes (PMTs), multiple windowPMTs, position-dependant wire detectors, position/time-sensitivedetectors, a photodiode array, an intensified photodiode array,charge-coupled devices (CCDs), intensified CCDs, an SIT or other videocamera, or any other suitable optical-to-electrical transducer.

In any event, the detector array 42 is preferably a linear array, withthe respective wavelength bands being incident upon a corresponding oneof the detectors 43. Each detector 43 in the array receives the lightincident upon it and generates a corresponding analog electrical signal.The electrical signals are then introduced to respectiveanalog-to-digital converters (shown schematically at 44) which convertthe incoming analog signals into corresponding digital signals. Thedigital signals are then delivered to a processor 46 that processes thedigital signals to determine the constituents of the emissions spectra,as is described in more detail below.

Referring now to FIG. 3, there is shown a system 50 according to analternative embodiment of the invention. In place of spectral analyzer28, system 50 includes a tunable filter 52 that is interposed betweenlens 24 and a single detector 54, for example, a PMT. In one embodiment,tunable filter comprises a liquid crystal tunable filter (LCTF) thatutilizes liquid crystals to continuously vary the retardance ofindividual filter stages, resulting in a narrow band filter that iselectrically tunable over a wide spectral range. Alternatively, thetunable filter 52 may comprise an acousto-optical tunable filter. In anyevent, tunable filter is controlled by a suitable control unit 56 tovary the bandpass of the filter through the spectral range. At eachbandpass, detector 54 receives fluorescence and generates acorresponding electrical output signal, which is converted to digitalformat by an analog-to-digital converter 58 and then introduced toprocessor 46 for processing.

Referring now to FIG. 4, there is shown a system 60 according to stillanother embodiment of the invention. System 60 includes a long passdichroic mirror 62 placed in the light stream between the scanningoptics 20 and the microscope lens 22. The dichroic mirror 62 is selectedsuch that wavelengths corresponding to the fluorescence radiated by thespecimen are reflected by the mirror 62, while the laser light fromlaser 14 passes through without being reflected. The reflectedfluorescence is directed to a focusing lens 64, and then introduced tolight guide 26 which delivers the light to spectral analyzer 28. Asdescribed above, spectral analyzer disperses the light and passes thelight on to ADC 44, which converts the respective bands into digitalsignals and introduces the digital signals to processor 46.

System 60 is therefore suitable for use in connection with anon-descanned two-photon microscope. By diverting the radiatedfluorescence before it passes through the scanning optics 20, a signalof increased intensity is received by spectral analyzer 28, as comparedwith a signal that passes through scanning optics 20 and dichroic mirror18 before being received by a spectral analyzer or detector. Thus, thedwell time at each pixel can be reduced as a result.

Referring now to FIG. 5, a system 70 is shown according to anotherembodiment of the invention. System 70 includes a light collector 72that substantially surrounds specimen 12 and includes a reflective innersurface. Thus, light emitted by fluorescent dyes within specimen 12 arecollected by the collector, regardless of the direction in which thelight radiates. In one embodiment, collector 72 comprises an integratingsphere. Alternatively, collector 72 may be in the shape of an ellipsoidor other structure that substantially encompasses specimen 12 to collecta substantial amount of the light radiating from specimen 12, forexample, an elliptical mirror. Collector 72 connects to light guide 26to deliver the collected light to spectral analyzer 28.

Thus, in use of the various systems described above, laser light isdirected by dichroic mirror 18, scanning optics 20, and focusing lens 22to a region of specimen 12. The photons (either from a single-photonlaser or from a multi-photon laser) excite the fluorescent dyes in theregion, causing them to fluoresce. The entire emitted spectrum isreceived and processed simultaneously in certain of the illustrativeembodiments to speed up the collection process. The spectral informationis then processed to determine the amounts of each dye contributing tothe emitted spectrum.

Referring now to FIG. 6, operation of processor 46 is described ingreater detail. In one embodiment, processor 46 is programmed to executea linear unmixing operation upon the incoming spectral data toapproximate the quantities of each fluorescent dye that contributed tothe emitted spectrum. As shown in FIG. 6, operation begins at step 100by determining spectral characteristics for the respective individualfluorescent dyes. As is well known in the art, each of the fluorescentdyes emits a particular spectrum over a certain wavelength band and atcertain varying intensities within that band (see FIGS. 7 a-c whichillustrate examples of emissions spectra for three different fluorescentdyes). At step 102, data relating to the probe spectra is recorded inprocessor memory for subsequent retrieval.

At step 104, processor 46 receives the measured imaging spectrum datafrom ADC 44 (see FIG. 7 d), and retrieves the characteristic spectra forthe various fluorescent dyes. At step 106, the measured spectrum isdecomposed into the various component dye spectra. This can beaccomplished in many different ways. In one embodiment, processor 46generates a model spectrum from the individual templates, and comparesthe model spectrum with the actual recorded spectrum. The respectiveweights of each of the individual templates are then varied to arrive ata close approximation of the actual spectrum. Then, at step 108,processor 46 determines the weights of each dye to quantitate therespective dye intensities.

In another embodiment, processor 46 is programmed to carry out principalcomponent analysis (PCA) on the incoming data. As is well known in theart, PCA is a linear model which transforms the original variables of anemission spectrum into a set of linear combinations of the originalvariables called principal components, that account for the variance inthe original data set. Suitable forms of PCA algorithms are disclosed inU.S. Pat. Nos. 5,991,653 to Richards-Kortum et al., and 5,887,074 to Laiet al., the disclosures of which are hereby expressly incorporated byreference.

From the foregoing, it will be apparent to those skilled in the art thatthe present invention provides an efficient and reliable system forreceiving and processing emissions spectra in connection withfluorescence microscopy. The system processes the emissions spectra todetermine concentrations of plural fluorescent dyes in a particularspectrum, even where the wavelength bands of the dyes overlap.

While the above description contains many specific features of theinvention, these should not be construed as limitations on the scope ofthe invention, but rather as one exemplary embodiment thereof. Manyother variations are possible. Accordingly, the scope of the inventionshould be determined not by the embodiments illustrated, but by theappended claims and their legal equivalents.

1. A system, comprising: a single photon laser configured to emitradiation capable of exciting a plurality of different excitable markersin a region of a cellular specimen to cause the plurality of differentexcitable markers to radiate florescence; a focusing lens configured tofocus radiation emitted by the single photon laser on the region of thecellular specimen; a dichroic mirror configured to reflect thefluorescence; a grating configured to separate the reflectedfluorescence into wavelength bands of fluorescence; a linear array ofhigh gain photomultiplier tubes configured so that each of thewavelength bands of fluorescence is incident upon a corresponding one ofthe high gain photomultiplier tubes and so that each of the high gainphotomultiplier tubes generates a corresponding analog signal; aplurality of digital to analog converters, each digital to analogconverter being configured to convert one of the analog signals from thelinear array of high gain photomultiplier tubes to a digital signal; anda processor configured to linearly unmix the digital signals.
 2. Thesystem of claim 1, wherein the dichroic mirror is configured so thatradiation from the single photon laser that is reflected from thespecimen passes through the dichroic mirror.
 3. The system of claim 1,wherein the processor is configured to identify the contribution to thefluorescence from each of the plurality of excitable markers.
 4. Asystem, comprising: a single photon laser microscope operative to causea plurality of different excitable markers in a region of a cellularspecimen to radiate fluorescence; photomultiplier tubes, each of theplurality of photomultiplier tubes being configured to receive acorresponding wavelength band of the florescence so that each of thephotomultiplier tubes generates a corresponding analog signal; aplurality of digital to analog converters, each digital to analogconverter being configured to convert one of the analog signals from thelinear array of high gain photomultiplier tubes to a digital signal; anda processor configured to unmix the digital signals.
 5. The system ofclaim 4, wherein the single photon laser microscope comprises a singlephoton laser configured to emit the radiation.
 6. The system of claim 5,further comprising a focusing lens configured to focus the radiation tothe region of the cellular specimen.
 7. The system of claim 4, furthercomprising a dichroic mirror configured to reflect the fluorescence. 8.The system of claim 7, wherein the dichroic mirror is configured so thatradiation from the single photon laser that is reflected from thespecimen passes through the dichroic mirror.
 9. The system of claim 4,further comprising a grating configured to separate the fluorescenceinto wavelength bands of fluorescence.
 10. The system of claim 4,wherein the photomultiplier tubes form a linear array.
 11. The system ofclaim 10, wherein the photomultiplier tubes are high gainphotomultiplier tubes.
 12. The system of claim 4, wherein thephotomultiplier tubes are high gain photomultiplier tubes.
 13. Thesystem of claim 4, wherein the processor is configured to linearly unmixthe digital signals.
 14. The system of claim 4, wherein the processor isconfigured to identify the contribution to the fluorescence from each ofthe plurality of excitable markers.
 15. A method, comprising: using thesystem of claim 1 to monitor cellular activity in a cellular specimen.16. A method, comprising: using the system of claim 3 to monitorcellular activity in a cellular specimen.
 17. A method, comprising:applying a plurality of different excitable markers to a cellularspecimen; applying light to the cellular specimen from a single-photonlaser microscope to cause fluorescence to be radiated from the region bymarkers in that region of the cellular specimen; using a grating toseparate the fluorescence into wavelength bands of fluorescence; andusing a linear array of photomultiplier tubes to detect the florescence,each photomultiplier tube receiving one of the wavelength bands offluorescence and generating a corresponding analog signal; convertingthe analog signals to digital signals; and linearly unmixing the digitalsignals.
 18. The method of claim 17, further comprising identifying thecontribution to the fluorescence from each of the plurality of excitablemarkers.
 19. A method, comprising: applying light from a single-photonlaser to a region of a cellular specimen having a plurality of differentexcitable markers, thereby causing florescence to be radiated from theregion of the cellular specimen; separating the fluorescence intowavelength bands of florescence using a grating; and detecting thefluorescence via photomultiplier tubes, each photomultiplier tubereceiving one of the wavelength bands of florescence and generating acorresponding analog signal; converting the analog signals to digitalsignals; and linearly unmixing the digital signals.
 20. The method ofclaim 19, further comprising applying the plurality of differentexcitable markers to the cellular specimen.
 21. The method of claim 19,wherein the single photon laser is part of a single photon lasermicroscope.
 22. The method of claim 19, wherein the photomultipliertubes form a linear array.
 23. The method of claim 22, wherein thephotomultiplier tubes are high gain photomultiplier tubes.
 24. Themethod of claim 19, wherein the photomultiplier tubes are high gainphotomultiplier tubes.
 25. The method of claim 19, further comprisingidentifying the contribution to the fluorescence from each of theplurality of excitable markers.
 26. A system, comprising: a singlephoton laser configured to emit radiation capable of exciting aplurality of different excitable markers in a region of a cellularspecimen to cause the plurality of different excitable markers toradiate fluorescence; a focusing lens configured to focus radiationemitted by the single photon laser on the region of the cellularspecimen; a dichroic mirror configured to reflect the fluorescence; agrating configured to separate the reflected fluorescence intowavelength bands of fluorescence; a linear array of high gainphotomultiplier tubes configured so that each of the wavelength bands offluorescence is incident upon a corresponding one of the high gainphotomultiplier tubes and so that each of the high gain photomultipliertubes generates a corresponding analog signal; a plurality of digital toanalog converters, each digital to analog converter being configured toconvert one of the analog signals from the linear array of high gainphotomultiplier tubes to a digital signal; and a processor configured toidentify the contribution to the fluorescence from each of the pluralityof excitable markers.
 27. A system, comprising: a single photon lasermicroscope operative to cause a plurality of different excitable markersin a region of a cellular specimen to radiate fluorescence;photomultiplier tubes, each of the plurality of photomultiplier tubesbeing configured to receive a corresponding wavelength band of thefluorescence so that each of the photomultiplier tubes generates acorresponding analog signal; a plurality of digital to analogconverters, each digital to analog converter being configured to convertone of the analog signals from the linear array of high gainphotomultiplier tubes to a digital signal; and a processor configured toidentify the contribution to the fluorescence from each of the pluralityof excitable markers.